The principle objective of this proposal is to understand the mechanism and secondary modulatory properties of the cardiac sodium-calcium (Na/Ca) exchange process. The experimentation is planned so as to establish a cohesive, integrated picture of exchanger function from the level of partial reactions of the exchange cycle up to the level of exchanger interactions with the sarcolemmal membrane. The experimentation will specifically investigate 1) the biophysical basis and kinetics of electrogenic reactions in the transport cycle, 2) the detailed function of secondary modulation ('gating') reactions and their properties in intact myocytes, 3) the functional properties of selected mutants of the cloned exchanger, 4) interactions of the exchanger with its lipid and cytoskeletal environment, and 5) the mechanism by which cytoplasmic MgATP stimulates exchange activity, in particular the possible role of aminophospholipid translocase. The proposed work in each of these areas will exploit innovative experimental techniques developed in this lab, in particular the giant excised membrane patch method (15-40 mum diameter patches; 5-18 pF; 1-10 Gomego seals). Studies of the partial electrogenic reactions of the exchanger will exploit the speeds of voltage clamp (ap. 5 mus) and solution switching (ap. 10 ms) which can presently be achieved in giant patches. To investigate the mechanisms for profound effects of phospholipid composition on exchange current, membrane phospholipid composition of giant patches will be modified during individual experiments by a novel method to transfer phospholipids to giant patches via the hydrophobic pipette coat. Optical methods employing fluorescent 'membrane probes' will be used to test the hypothesis that stimulation of cardiac exchange activity by MgATP involves the establishment of phosphatidylserine (PS) asymmetry by an aminophospholipid translocase. It may be expected that this work will progress toward a comprehensive understanding of the Na/Ca exchange process, toward more adequate methodologies in the study of membrane transport, and toward an understanding of physiologically important regulatory mechanisms in the control of transport function. The knowledge to be gained is fundamental to an understanding of cardiac function in both physiological and pathological settings.
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